Automated fraction re-analysis does it really make sense? Application. Udo Huber. Abstract

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1 Automated fraction re-analysis does it really make sense? Application Udo Huber mau 3500 Yellow Red Blue Pink min Abstract Today preparative HPLC is the most prominent technique for the purification of compounds in drug discovery. Due to the availability of completely automated, preparative HPLC systems equipped with mass-selective detector (MSD) it is the only technique fast enough for the purification of compounds from high-throughput synthesis. As a result of highthroughput purification large number of fractions have to be re-analyzed to check their purity before the target compounds are released for activity testing. The logical consequence would be a HPLC system that can not only purify but also automatically re-analyze the fractions directly after collection. In this Application Note we discuss automated fraction reanalysis and examine if the gain in automation is really worth the risks involved with this feature.

2 Introduction With the introduction of the purification system based on the 220 microplate sampler in 1998 Agilent offered a system that was not only capable of purifying compounds but also to automatically re-analyze the collected fractions and check them for purity 1. With the new Agilent 1100 Series purification system 2,3 automated fraction re-analysis is not possible due to the different design: the autosampler and fraction collector are no longer a single module but two separated, dedicated modules for injection and fraction collection. In this Application Note we will share some inputs from Agilent purification system users, which explain why it is better to perform the re-analysis for purity checks not directly after fraction collection but at a later stage in the sample workflow on a dedicated analytical HPLC system. Equipment The experiments shown in the section Concentration gradient (figure 3) were performed on an Agilent 1100 Series system containing the following modules: Two Agilent 1100 Series preparative pumps Agilent 1100 Series preparative autosampler Agilent 1100 Series column organizer Agilent 1100 Series diode array detector Agilent 1100 Series fraction collector PS The system was controlled using the Agilent ChemStation (rev. A.09.03) and the Purification/High- Throughput software (rev. A.01.02). Results and discussion Typical workflow The typical workflow in drug discovery but also in other industries searching for active compounds, for example Crop Science, can be described in six steps: 1. Submission of the impure sample by the chemist. Very often the sample solvent is not identical with the mobile phase used for preparative HPLC but has to be a strong solvent such as DMSO or DMF. 2. Purification run on the preparative HPLC system, often by reversed phase chromatography using water and acetonitrile or methanol as mobile phase. 3. Fraction collection. 4. Solvent evaporation and weighing of the compound. 5. Re-dissolving the pure compound to a certain concentration, often again in a strong solvent like DMSO or DMF. 6. Submission of a small portion of the solution for activity testing, rest of the sample goes to storage. At a certain point in this workflow it has to be checked that the compound submitted for activity testing (step 6) has the required purity, which must usually be higher then %. Therefore, a representative portion of the sample that goes for activity testing has to be re-analyzed. Using automated fraction re-analysis this would be done directly after fraction collection (step 3) directly from the fraction container. Based on customer experience we recommend to do the purity check of the sample right before submission for activity testing (step 6). Disadvantages of automated fraction re-analysis When doing automated fraction re-analysis the sample for measuring the purity of the compound is taken directly after fraction collection (step 3). The rest of the fraction has to be evaporated and redissolved before the compound goes for activity testing (step 6). The problem is that the sample drawn directly from the fraction is not really a representative portion of the sample that goes for activity testing, because the following errors can occur: 1. Concentration gradient 2. Crystallization 3. Decomposition during evaporation 1. Concentration gradient The target compound elutes from the column over a certain time leading to a concentration gradient in the fraction. When the fraction start is triggered the compound concentration in the mobile phase is rather low. It increases up to the highest concentration at the peak apex and decreases again until the peak end is triggered. To demonstrate this effect several dyes were injected onto a column and a fraction was collected based 2

3 on a retention time window. The result is shown in figure 1. The concentration gradient of the two dyes can be seen clearly in the fraction container. If a small volume is drawn directly from the tube for re-analysis this sample is certainly not representative for the sample submitted for activity testing. It could happen, for example, that the concentration of a co-eluting byproduct in the drawn volume has a much higher concentration than it would have in the wellmixed sample (figure 2). Two questions arise when looking at the concentration gradients in figures 1 and 2: Will the fraction be mixed completely when collecting a higher flow rates and how long does it take to get a homogenous solution due to diffusion? Therefore a series of measurements, also using dyes, where performed. The results are shown in figure 3. It can be seen that even at higher flow rates (20 ml/min) no mixing of the fraction occurs while it is collected. It can also be seen that even after 24 hours there is still a concentration gradient in the fraction, there is no complete mixing due to diffusion. Those results clearly show that a sample drawn directly from the fraction container is never a representative portion of the complete fraction, even if the re-analysis is done a few hours after the fraction was collected. mau Yellow Red Blue Figure 1 Concentration gradient in the fraction container Pink min Sample drawn for re-analysis. Is this representative? Blue Red Figure 2 Error due to concentration gradient. 3

4 2. Crystallization For good solubility, samples are very often submitted for purification not in the solvents used as mobile phase but in strong solvents like DMSO or DMF. However, the mobile phase used for reversed phase chromatography is usually water and acetonitrile or methanol. This means the collected fraction contains the target compound in a water/acetonitrile or water/methanol mixture. Depending on the injected sample amount and solubility that the target compound starts to crystallize out of the mobile phase (figure 4). If a sample volume for the purity check is drawn from the mother liquid this sample is again not representative for the sample going for activity testing. If, for example, there is, an impurity in the fraction that does not crystalize, the concentration of this impurity appears to be higher then it really is. On the other hand, if the impurity crystallizes out, the purity of the target compound appears to be higher than it really is. T = 0 h T = 6 h T = 24 h Figure 3 Mixing effects in dye fractions. T = 96 h Flow = 1ml/min Flow = 4.5ml/min Flow = 20ml/min 3. Decomposition during evaporation After purification the mobile phase of the fraction containing the target compound has to be evaporated. This can be done either in a vacuum centrifuge or by heating and flushing with nitrogen. After weighing the residue it has to be re-dissolved to a certain concentration using an appropriate solvent, for example, DSMO or DMF. If the purity check of the target compound was done directly from the fraction container a possible decomposition of the target compound during the evaporation Figure 4 Crystallization from mobile phase Sample drawn for re-analysis. Is this representative? 4

5 process would not be recognized (figure 5). In the worst case it could happen that a highly active compound could be missed. If the re-analysis and purity check of the target compound directly from the fraction container confirms the target mass and shows good purity but the activity test of the decomposed compound shows no activity a possible drug candidate was lost. It was also not recognized that the compound, on which the successful reanalysis was performed and the compound that went for activity testing were not the same. While a second reanalysis is always done for a compound that shows activity this is not the case for an inactive compound. Evaporation Sample drawn for re-analysis. Is this representative? Figure 5 Sample decomposition during evaporation Sample decomposes while evaporating Dissolved in DMSO Portion taken to activity testing Which is the right step to perform the purity re-analysis? The three problems described in the previous section do not occur when the reanalysis is not performed directly after fraction collection but at a later step in the workflow. Since a portion of the re-dissolved compound goes for activity testing (step 6) a second portion of the same solution could be used for analysis and purity testing (figure 6). This makes absolutely sure that a representative sample of the compound going for activity testing is reanalyzed. Since the fraction had to be evaporated and re-dissolved before a portion is taken for activity testing it makes perfectly sense to carry out the purity check on an analytical HPLC system. Even if the target compound had decomposed during evaporation this was recognized because the re-analysis would show the wrong target mass. Sample drawn for re-analysis. This is representative! Figure 6 Representative sample for purity testing Portion taken to activity testing 5

6 Conclusion Automated fraction reanalysis would be another step towards a completely automated purification solution but in this Application Note we showed that the gain in automation sacrifices the reliability of the measured results. We did not only show that the measured purity can lead to wrong results due to the concentration gradient and possible crystallization in the fraction container but also that an active compound could be completely missed due to decomposition during evaporation. All this disadvantages can be overcome if the analysis is performed at a later step of the sample purification and activity testing process on a dedicated analytical HPLC system. This does also not tie up the valuable purification system for routine analytical work. Automated fraction reanalysis might add some automation to the purification process but at the risk of unreliable or completely wrong results. References 1. Agilent 1100 Series purification system: Fractionation and automated re-analysis of fractions, Agilent Technologies Application Note, publication number EN, New dimensions for HPLC applications, Agilent Technologies Brochure, publication number EN, Agilent 1100 Series Purification Platform Customized system configurations, Agilent Technologies Flyer, publication number EN,

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8 Udo Huber is Application Chemist at Agilent Technologies, Waldbronn, Germany. The information in this publication is subject to change without notice. Copyright 2003 Agilent Technologies All Rights Reserved. Reproduction, adaptation or translation without prior written permission is prohibited, except as allowed under the copyright laws. Published April 1, 2003 Publication Number EN